DNA analysis by hybridization is a known method in molecular biology (cf. “Gentechnische Methoden”, [“Genetic Engineering Methods”], G. Gassen and G. Schrimpf, Spektrum Akademischer Verlag Heidelberg, 1999, pages 243 to 261). This technique plays an important part in the detection of specific nucleic acids, e.g. in the molecular diagnosis of single point mutations (single nucleotide polymorphism, SNP). In this case, a probe oligonucleotide comprising a sequence of e.g. approximately 20 nucleotides is used to bind to nucleic acids that differ only in a single nucleotide. It is noted that in the present context the expression “nucleic acids” is intended to encompass a nucleic acid sequence, e.g. a DNA sequence or RNA sequence. Given a suitable choice of the hybridization conditions (in particular temperature and salt concentration), the probe oligonucleotide selectively binds the non-mutated variant of the nucleic acid, while the nucleic acid variant having the single point mutation does not bind, or binds only weakly. Detection of single point mutations is thereby possible. On account of the small differences in terms of binding energy between the variant without a mutation (that is to say the wild type) and the mutant, the reaction conditions with regard to temperature and also composition and salt concentration of the reaction solution have to satisfy exact stipulations.
Since the corresponding nucleic acids in the sample material (e.g. blood) are usually not available in sufficient quantity or concentration, it is necessary to amplify the nucleic acids to be examined. This amplification can be effected in a sequence-specific manner by various methods known in molecular biology, e.g. by SDA (strand displacement amplification), described in Walker, G T, et al., “Strand Displacement Amplification, an isothermal, in vitro DNA Amplification Technique”, Nucleic Acids Research, 1992, 20, 1961 to 96; by TMA (transcription mediated amplification), described in www.gen-probe.com/sci_tech/tma.htm; or by polymerase chain reaction (PCR), described in U.S. Pat. No. 4,683,195, inter alia. One problem here is that the composition of the reaction solution of the amplification reaction, and hence the “amplification crude product”, does not have the composition, and in particular salt concentrations, required for hybridization. For molecular diagnosis, it may additionally be necessary to selectively separate the hybrids formed in a subsequent process (melting) e.g. by increasing the temperature. In order to enable hybridization processes with a high yield, a high concentration of monovalent cations (e.g. Na+ ions) is necessary, inter alia. Monovalent cations promote the formation of the double helix structure during the hybridization reaction.
However, the reaction mixtures of the amplification reactions, e.g. for a PCR reaction, contain a low concentration of monovalent cations. Furthermore, PCR reaction buffers have a relatively high concentration (a few mM) of Mg2+ ions, which, during the hybridization to detection probes, adversely affect the binding of probes and complimentary strands to form complete hybrids, can bring about an extension of the probes by polymerase activity and, during a subsequent melting process, bring about a stabilization of double strands and make melting more difficult, which leads to “washed out” melting curves at high temperatures.
In accordance with the prior art, amplification products are therefore purified before the hybridization reaction; in this case, all components that disturb a hybridization reaction (inter alia polymerase, primers, nucleotides, salts) are removed and the concentration of Na+ ions is increased. This purification process is relatively complicated and is usually effected by non-specific binding of the nucleic acids to a solid phase (by way of so-called purification columns), washing of the amplification product on the column and dissolution from the solid phase or by phenol/chloroform extraction or similar methods. Particularly when carrying out nucleic acid analyses in microfluidic devices, wherein all the reaction processes proceed in an integrated manner and in a small space, the purification methods that are customary in the prior art are not appropriate since their realization is too complicated, under these circumstances.